The invention discloses a separation and culture method of umbilical cord blood derived mesenchymal stem cells, and belongs to the technical field of biology. The separation and culture method comprises the following steps of performing centrifuging treatment on umbilical cord blood, taking an upper layer to obtain plasma, and taking a lower layer to obtain cells; performing inactivation treatmenton the plasma, then performing centrifuging treatment, and taking supernatant so as to obtain autoserum; mixing the lower-layer cells with normal saline to obtain a mixture, then placing the mixturein lymphocyte separation liquid, and performing centrifuging treatment so as to obtain umbilical cord blood mononuclear cells; and re-suspending the umbilical cord blood mononuclear cells with serum-free culture mediums, placing the re-suspended umbilical cord blood mononuclear cells in an autoserum coating culture dish, performing primary culture to obtain primary mesenchymal stem cells, adding cell dissociation liquid into the primary mesenchymal stem cells, performing cell dissociation treatment, and then performing subculturing. According to the method, penicllin-streptomycin for cell culture is not added in a separation process and a culture process, so that toxicity is not generated on the cells. In addition, according to the method, the autoserum coating culture dish for centrifugalseparation is adopted, so that animal derived viruses cannot be introduced.